Name: monitoring kinase and phosphatase activities through the cell cycle by ratiometric fret
Uploaded: 2012-01-27T12:00:00
Description: Watch this Scientific Journal Video about Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET at JoVE.com

Forfatterne er støttet av det svenske forskningsrådet, den svenske Stiftelsen for strategisk forskning, den svenske kreft samfunnet, svenske barn Kreftforeningen, Åke Wibergs fundament og Jeanssons fundament.

1. Introduserer sonden til celler <ol><li> Co-transfektere celler med en FRET-basert probe og en plasmid overdragelse motstand. Velg et transfeksjon metode som er effektiv i din celle-type av interesse. For U2OS celler, standard kalsiumfosfat transfeksjon metodene gir tilfredsstillende resultater 15. </li><li> Merk cellene i riktig antibiotika i minst sju dager. Dette beriker mengden av celler uttrykker sonden og begrenser mengden av celler med giftige uttrykk nivåer, eller uttrykk nivåer som sterkt påvi...

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Overvåking FRET gjennom cellesyklus krever hensyn som er mindre avgjørende ved vurderingen av kortsiktig respons på ytre stimuli. Først er cellesyklus progresjon lett opprørt av stress-signalering, som krever at fototoksisitet holdes til et minimum. Sekund, kan alle reportere potensielt påvirke cellulære prosesser ved titrering ut kinaser, fosfataser eller interaksjon domener. Den sannsynligvis enkleste måten å vurdere om den eksperimentelle forholdene er tilstrekkelige, er å måle cellen syklus lengde fra mit...

<table border="1"><tbody><tr><td> Reagens </td><td> Katalog nummer </td><td> Firma </td></tr><tr><td> Leibovitz L-15, ingen fenol rødt </td><td> 21083-027 </td><td> GIBCO, fra Life Technologies </td></tr><tr><td> DMEM + Glutamax-I </td><td> 31966 </td><td> GIBCO, fra Life Technologies </td></tr><tr><td> Føtal Bovine Serum (FBS) </td><td> SV30160.03 </td><td> HyClone </td></tr><tr><td> 0,05% Trypsin EDTA </td><td> SH30236.01 </td><td> HyClone </td></tr><tr><td> Penicillin-Streptomycin </td><td> SV30010 </td><td> HyClone </td></tr><tr><td> DPBS </td><td> 14287 </td><td> GIBCO, fra Life Technologies </td></tr><tr><td> Puromycin </td><td> P8833 </td><td> Sigma-Aldrich </td></tr></tbody></table>

monitoring kinase and phosphatase activities through the cell cycle by ratiometric fret

Förster resonans energi overføring (FRET)-baserte journalister en tillate vurdering av endogene kinase og fosfatase aktiviteter i levende celler. Slike prober typisk bestå av varianter av CFP og YFP, grepet av en phosphorylatable sekvens og en fosfor-bindende domene. Ved fosforylering, sonden endringene konformasjon, som resulterer i en endring av avstand eller retning mellom CFP og YFP, fører til en endring i FRET effektivitet (fig 1). Flere sonder har blitt publisert i løpet av det siste tiåret, overvåke aktiviteten balansen av flere kinaser og fosfataser, inkludert journalister på 2 PKA, 3 PKB, 4 PKC, 5 PKD, 6 ERK, 7 JNK, 18 CDK, Aurora B 9 og Plk1 9 . Gitt den modulære design, flere prober er sannsynlig å dukke opp i nær fremtid 10. Progresjon gjennom cellesyklus er påvirket av stress signaling veier 11. Spesielt er cellesyklus reguleres annerledes under uforstyrret vekst i forhold til når cellene er utvinne fra stress 12. Time-lapse avbildning av celler gjennom cellesyklus krever derfor spesiell forsiktighet. Dette blir et problem spesielt ved ansettelse ratiometric imaging, siden to bilder med høyt signal til støyforhold er nødvendig for å tolke resultatene. Ratiometric FRET avbildning av cellesyklus avhengige endringer i kinase og fosfatase aktiviteter har hovedsakelig vært begrenset til sub-seksjoner av cellesyklus 8,9,13,14. Her diskuterer vi en metode for å overvåke FRET-baserte sondene bruker ratiometric bildebehandling hele den menneskelige cellesyklus. Metoden baserer seg på utstyr som er tilgjengelig for mange forskere i life sciences og krever ikke ekspertkunnskap om mikroskopi eller bildebehandling.

Watch this Scientific Journal Video about Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET at JoVE.com

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

FRET-baserte journalister blir stadig mer brukt til å overvåke kinase og fosfatase aktiviteter i levende celler. Her beskriver vi en metode om hvordan du bruker FRET-baserte journalister å vurdere celle syklus-avhengige endringer i mål fosforylering.

Overvåking Kinase og fosfatase Aktiviteter Gjennom Cell Cycle av Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

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